Biochemistry 2009, 48, 3219–3229
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Allosteric Effect of Amphiphile Binding to Phospholipase A2‡ Bao-Zhu Yu,§ Shi Bai,§ Otto G. Berg,*,| and Mahendra K. Jain*,§ Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716, and Department of Molecular EVolution, Uppsala UniVersity EVolutionary Biology Center, Uppsala, Sweden ReceiVed July 2, 2008; ReVised Manuscript ReceiVed December 30, 2008
ABSTRACT:
In the preceding paper, we showed that the formation of the second premicellar complex of pig pancreatic IB phospholipase A2 (PLA2) can be considered a proxy for interface-activated substrate binding. Here we show that this conclusion is supported by results from premicellar Ei# (i ) 1, 2, or 3) complexes with a wide range of mutants of PLA2. Results also show a structural basis for the correlated functional changes during the formation of E2#, and this is interpreted as an allosteric T (inactive) to R (active) transition. For example, the dissociation constant K2# for decylsulfate bound to E2# is lower at lower pH, at higher calcium concentrations, or with an inhibitor bound to the active site. Also, the lower limits of the K2# values are comparable under these conditions. The pH-dependent increase in K2# with a pKa of 6.5 is attributed to E71 which participates in the binding of the second calcium which in turn influences the enzyme binding to phosphatidylcholine interface. Most mutants exhibited kinetic and spectroscopic behavior that is comparable to that of native PLA2 and ∆PLA2 with a deleted 62-66 loop. However, the ∆Y52L substitution mutant cannot undergo the calcium-, pH-, or interface-dependent changes. We suggest that the Y52L substitution impairs the R to T transition and also hinders the approach of the Michaelis complex to the transition state. This allosteric change may be mediated by the structural motifs that connect the D48-D99 catalytic diad, the substrate-binding slot, and the residues of the i-face. Our interpretation is that the 57-72 loop and the H48DNCY52 segment of PLA2 are involved in transmitting the effect of the cooperative amphiphile binding to the i-face as a structural change in the active site. Premicellar Ei# (i ) 1, 2, or 3) complexes of pig pancreatic IB phospholipase A2 (PLA2)1 are viable surrogates for some of the structural and functional states of the interfaceactivated E* form (1-5). Ei# complexes have stoichiometry of Ni amphiphiles bound per enzyme with Hill numbers ni and dissociation constant Ki#. These parameters provide a measure of multiple amphiphile interactions with the i-face. As shown in the preceding paper (5), the affinity of the inhibitor for Ei# complexes is greater than that for the E form. It is analogous to the KS* activation (6, 7) at the interface. Results in this paper show that the structural effects on the enzyme are responsible for an allosteric change by which a conformation change associated with the binding of amphiphiles to form E2# is correlated with enhanced inhibitor binding to the active site and, conversely, binding at the ‡ This work is dedicated to Bert Verheij (January 20, 1944, to August 1, 1998) who shared thoughts on PLA2. * To whom correspondence should be addressed. Telephone: (302) 831-2968. Fax: (302) 831-6335. E-mail:
[email protected]. § University of Delaware. | Uppsala University Evolutionary Biology Center. 1 Abbreviations: CMC, critical micelle concentration; DC7PC, 1,2diheptanoylphosphatidylcholine; DMPM, dimyristoylphosphatidylmethanol; HDNS, N-dansyl-hexadecylphosphoethanolamine; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; i-face, interface binding surface of PLA2; MJ33, 1-hexadecyl-3-(trifluoroethyl)-rac-glycero-2phosphomethanol; PCU, 2-dodecanylaminohexanol-2-phosphocholine; PLA2, secreted type IB phospholipase A2 from pig pancreas; ∆PLA2, 62-66 loop deleted PLA2; ∆Y52L, ∆PLA2 with the Y52L substitution; RET, fluorescence resonance energy transfer; STD, saturation transfer difference proton NMR; TMA-DPH, trimethylammonium diphenylhexatriene.
active site promotes a conformation with enhanced amphiphile binding to the interface. Verheij and co-workers designed and characterized functional properties of a wide range of PLA2 mutants (8-16). These and other results (1, 16-20) are consistent with the key requirement of the interfacial kinetic paradigm for PLA2 (21) that the active site be buried inside the structure and that the i-face be on the surface of PLA2. These results also suggest coupling between the i-face and active site events mediated by the structural features of PLA2, including the 57-71 loop and the highly conserved H-bonding network (Figure 1). We characterize the premicellar complexes of the PLA2 mutants designed by Verheij et al. The key result is that decylsulfate binding to the i-face of E2# is enhanced in most mutants to the same limiting value at low pH, at high calcium concentrations, or with an inhibitor bound to the active site. These effects on K2# are not observed with the ∆Y52L mutant with the Y52L substitution in the 62-66 loop deleted PLA2, and the results show that it remains frozen in one form. In the Appendix, the structural coupling between the i-face and active site is modeled in terms of an allosteric T (inactive) to R (active) transition, where the R state is the form that binds well at both the i-face and the active site. The results suggest that the Y52L substitution in ∆PLA2 confers a conformation that is permanently locked in the R form. EXPERIMENTAL PROCEDURES The research group of the late B. Verheij (Utrecht, The Netherlands) provided the mutants and PCU for this study.
10.1021/bi801245s CCC: $40.75 2009 American Chemical Society Published on Web 03/20/2009
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Yu et al. Scheme 2
dependence were acetate (pH 4-5), citrate (pH 5.5-6.5), HEPES (pH 6.5-7.5), Tris (pH 7.5-8.5), and borate (pH 9-10). The CMC of decylsulfate is 4.40 mM in the pH 6.9 buffer containing 0.48 M NaCl compared to a CMC of 4.50 in the absence of added NaCl (17). Decylsulfate titration curves were fitted as described below. The stepwise change in the Trp-3 emission intensity with the increasing concentration, cf, of monodisperse decylsulfate below the CMC is analytically described as δF ) ((cf ⁄ K1#)n1{a1 + (cf ⁄ K2#)n2[a2 + a3(cf ⁄ K3#)n3]}) ⁄ (1 + (cf ⁄ K1#)n1{1 + (cf ⁄ K2#)n2[1 + (cf ⁄ K3#)n3]}) (1) FIGURE 1: Hydrogen bonding network of PLA2 (16, 32) that consists of the structural water (W1), the NH2 group of A1, the backbone oxygens of P68 and E71, the side chains of N4, Y52, and Y73, and the catalytic diad H48-D99(εNH). Calcium in the catalytic site is 7-coordinated to the carboxyl group of D49, backbone carbonyls of residues 28, 30, and 32, and two water molecules. The H48DNCY52 segment is localized on the 41-56 helix that links the 25-36 loop that binds the catalytic calcium and the 57-72 loop that binds the low-affinity calcium on the other side. Residues of these loops and the N- and C-terminal region provide side chains for the i-face interactions and allosteric coupling to the active site events via the H-bonding network.
Scheme 1
Interfacial kinetic behavior of the mutants on DMPM (22, 23) vesicles or DC7PC micelles (7) was characterized to ensure the integrity of the samples. The initial rates at 24 °C, pH 8.0, and a substrate mole fraction of 1 are given in the tables. The interfacial parameters expressed with an asterisk have the same meaning as those for the solution enzymes (21, 24). Other reagents, methods, protocols, and precautions used to characterize the binding of monodisperse amphiphiles to the i-face and the active site are as described previously (1, 2, 17). Decylsulfate Binding to PLA2. Established experimental protocols, theory, and interpretation of the decylsulfate binding model in Scheme 1 are used (1-3, 5, 17) with specific details given below or in the figure captions and text. Typically, the decylsulfate concentration-dependent change in Trp-3 fluorescence from 1 or 2 µM PLA2 was monitored on an SLM-Aminco AB2 instrument. It was set in the ratio mode with 4 nm slit widths with excitation at 280 nm and emission at 333 nm for the Trp signal, or at 450 nm for the RET signal from the Trp/TMA-DPH pair, or at 490 nm from the Trp/HDNS pair. Intensity values obtained with an integration time of 4 s have a noise level of 0.5 mole fraction both in the presence and in the absence of calcium (3). The low affinity of decylsulfate for the active site of Ei# permits study of the effect of inhibitors on the decylsulfate binding parameters (5). PCU with an sn-2-amide group is a
Table 1: Effect of Calcium (millimolar), pH, and 5 µM PCU on Decylsulfate Binding to PLA2a pH
[PCU]
[Ca]
K1#
K2#
8.0
0 0 0 5 µMb 0 0 0 5 µMb 0 0 5 µMb
eg 0.5 20 0.5 eg 0.5 20 0.5 0.5 20 0.5
0.04 0.12
1.35 1.1 0.38 0.5 0.79 0.76 0.25 0.43 0.15 0.16 0.12
K3#
a1
a2
a3
n1
n2
0.52
1.4 1.6
1.15 0.54 0.64
1 1.7 1.6
0.52 0.01
1.2 2.3 1.8 2
5 6 5 3.4 8 7 6 5 8 10 8
n3
Vo (s-1)
8
270
PLA2
6.9
4.0
0.07 0.033 0.08 0.047 0.02 0.03 0.02
5.2 2.5 2.9 3.0 2.47 2.3 2
0.12 0.18 0.05 0.10 0.24 0.28 0.27 0.10 0.12 0.14
0.49 0.54 0.75 1.02 0.58 0.67 0.73 0.72 0.80 0.75 0.72
0.41
3 6 8 5 4 1.4
H48Q 8.0 4.0
a
0 0 5 µMb 0 0 5 µMb
eg 0.5 0.5 eg 0.5 0.5
1, K3# ) 5.9, and n3 ) 0.7.
is apparent in a 4-fold pH effect on K2# for ∆PLA2 compared to the 7-fold pH effect on PLA2. Substitutions at positions 31, 53, 56, and 69 in ∆PLA2 had no additional effect on K2#, although charge compensation of Lys-53 and Lys-56 in PLA2 has a modest effect on the i-face interactions with the zwitterionic interface (3, 27, 38, 39). Substitution of Leu31 and Tyr-69 in ∆PLA2 had a small effect on decylsulfate binding, although both of these residues are in contact with the inhibitor in the active site (29, 40, 41). The decylsulfate binding parameters for the Trp substitution mutants (20) were also comparable to those of PLA2 with or without PCU, although ai values from Trp at positions 6, 10, 19, 20, and 31 are noticeably different (results not shown). The hydroxyl groups of Tyr-52 and Tyr-73 are part of the highly conserved H-bonding network (Figure 1) that is conserved in all secreted PLA2 forms (15, 26-28). The decylsulfate binding behaviors of the Y52 and Y73 substitution mutants of ∆PLA2 are particularly revealing. As summarized in Table 3, the Y to F substitution of one or both of these tyrosines in ∆PLA2 has at best a modest effect on any of the parameters. On the other hand, ∆Y52L (Table 4) and ∆Y73L (Table 3) are catalytically impaired. The Y to L substitution also has a significant effect on the Trp-3 emission peak intensities of the E form. Compared to the relative intensity of 1 for free PLA2, it is 1.5 for ∆Y73L, 0.75 for ∆Y52L, and 0.43 for ∆PLA2. Low ai values for the Ei# complexes of ∆Y73L precluded detailed analysis. Results described next show that the Y52L substitution in ∆PLA2 has a dramatic effect on decylsulfate binding and the associated functional changes. Anomalous Decylsulfate Binding to ∆Y52L. The decylsulfate binding behavior of ∆Y52L (Table 4) is significantly different compared to that of ∆PLA2 or any other mutant that we tested. The difference provides a useful insight into the coupling of the active site and i-face events. Decylsulfate binding curves of virtually all PLA2 and ∆PLA2 mutants showed three well-resolved steps with a significant effect of pH and PCU on K2#. However, results in Figure 5 show that the curves for ∆PLA2 and ∆Y52L are different at pH 4 and 8. Not only is the shape of the curves for ∆Y52L anomalous, but the effect of pH or PCU is hardly noticeable. The decylsulfate binding to ∆Y52L is fitted with two assumptions
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FIGURE 6: Decylsulfate concentration dependence of the relative quenching (F/Fq ratio): (O) PLA2 and (2) ∆Y52L mutant at pH 8.0 in 10 mM Tris, 10 mM NaCl, and 0.5 mM CaCl2. F is the emission intensity without and Fq the emission intensity with 0.15 M succinimide.
to obtain the parameters summarized in Table 4. First, the rising phase is due to E1# and E2# formation, and the falling phase is due to E3#. Results described below show clustering of bound decylsulfate with the formation of E2#. As it is difficult to imagine amphiphile clustering without any Hill cooperativity, this leads to the second assumption that E2# is “hidden” behind E1# (i.e., a2 ) a1), and therefore, K2# cannot be clearly resolved as in other cases where the two steps are well separated. This crucial assumption leaves n2 virtually indeterminate from the fit. Apparently, the first two steps are not resolved for ∆Y52L, as expected if there is no conformational change in the formation of E2#. Furthermore, the Trp-3 signal at 333 nm at the peak in the decylsulfate titration curve for ∆Y52L (F ) 45 at pH 8 and 0.5 mM calcium) is in fact higher than the peak [F ) 34 (cf. Figure 5)] for ∆PLA2. At the same time, the basal signal (in the absence of decylsulfate) is higher for ∆Y52L than for ∆PLA2 (F0 ) 27.3 vs 13.6) as if ∆Y52L even without decylsulfate present is closer to the conformation (R) with the larger signal. Clustering of Decylsulfate on ∆Y52L. The Hill numbers for most of the mutants in Tables 1-3 show cooperative clustering of many decylsulfate molecules around Trp-3 on the i-face. Since ni is expected to be smaller than the binding stoichiometry, Ni (eq 1), in most mutants a total of at least 2, 10, and 20 decylsulfate molecules are bound to E1#, E2#, and E3#, respectively. This is consistent with the result that the quencher accessibility of Trp-3 decreases for the higher Ei# complexes (2, 3) and E* (42). However, in spite of the apparent loss of Hill cooperativity, significant clustering occurs in ∆Y52L measured at sufficiently low enzyme and high decylsulfate ratios. As shown in Figure 6, Trp-3 is less quenched by succinimide in ∆Y52L than in PLA2. Also, for PLA2, an abrupt decrease occurs at >0.5 mM decylsulfate, whereas for ∆Y52L, the abrupt decrease is nearly complete at 0.1 mM decylsulfate. This difference is consistent with the estimated K2# values. The kinetics of covalent modification of ∆Y52L with N-bromosuccinimide is significantly slower at 0.1 mM decylsulfate (results not shown) which is expected if decylsulfate molecules clustered around Trp-3 shield access to the reagent (3, 20). Clustering of several decylsulfate molecules on ∆Y52L is also affirmed by the behavior of the RET probes partitioned in the Ei# complexes (2, 17, 43, 44). As shown in Figure 7, at low decylsulfate concentrations the shape of the titration curve with ∆Y52L is noticeably different compared to that
Yu et al.
FIGURE 7: Decylsulfate concentration (log scale) dependence of the RET signal (excitation at 280 nm, emission at 450 nm) from a mixture of 1 µM TMA-DPH with 1 µM ∆PLA2 (b) or ∆Y52L (O) at pH 8.0 in 10 mM Tris, 10 mM NaCl, and 0.5 mM CaCl2.
FIGURE 8: Decylsulfate concentration dependence (log scale) of the RET signal (excitation at 290 nm, emission at 490 nm) from a mixture of 1 µM HDNS and 1 µM ∆Y52L (2) or ∆PLA2 (9) at pH 8.0 in 10 mM Tris, 10 mM NaCl, and 0.5 mM CaCl2.
with ∆PLA2. The signal from ∆PLA2 and TMA-DPH is modestly quenched with the formation of E1# at 50 µM. Thus, a K0 of >500 is expected. Furthermore, the limiting value for K2#,app of 0.1-0.13 mM which appears for various mutants, at high PCU concentrations, at low pH, and/or at high calcium concentrations would correspond to K2#,R. Thus, K0 could be estimated from eq 5 as K0 ) [K2#,app(I ) 0)/0.13]n2 - 1; for PLA2 at pH 6.9, this gives K0 ∼ 104-105. At pH 4, the equilibrium for the free enzyme is pushed more toward the active R state and K0 is no longer .1, making the corresponding quantitation of KIR impossible. In the absence of I, K2#,R increases by a factor 1.4 for PLA2 when the pH increases from 6.9 to 8. If it is assumed that the pH effect in eq 5 resides only in R-T equilibrium constant K0 and not in K2#,R, K0 would be a factor 1.4n2 ≈ 9 larger at pH 8 than at pH 6.9; in other words, the increased pH pushes the R-T equilibrium even more toward T. For the ∆Y52L mutant (Table 4), K2#,app remains around 0.07 mM, independent of pH or the presence of PCU. In terms of eq 5, this implies K2#,R ) 0.07 mM and that K0 < 1; thus, the equilibrium is pushed toward R. In this limit, the binding to the active site as determined by KIR is indeterminate from the data and could also be influenced by the mutation. Alternatively, when the coupling is destroyed, the allosteric scheme is disrupted and the ∆Y52L mutant could have its i-face in the R conformation while the active site remains in something resembling the T conformation. However, the strong binding of PCU to E* (Table 5) suggests also that the active site of ∆Y52L remains in the R conformation. Although the numbers discussed above should be considered mostly as order-of-magnitude estimates, they show that the major effects and trends are consistent with the simplest possible concerted allosteric model as given by Scheme 3. The results of the previous paper suggest that PCU binding to the i-face can achieve partial activation. However, Scheme 3 does not account for the complication from PCU binding at the i-face, differences in n2 values, and possible partial conformation changes. #,app
REFERENCES 1. Yu, B. Z., Apitz-Castro, R., Tsai, M. D., and Jain, M. K. (2003) Interaction of monodisperse anionic amphiphiles with the i-face of secreted phospholipase A2. Biochemistry 42, 6293–6301.
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2. Berg, O. G., Yu, B. Z., Chang, C., Koehler, K. A., and Jain, M. K. (2004) Cooperative binding of monodisperse anionic amphiphiles to the i-face: Phospholipase A2-paradigm for interfacial binding. Biochemistry 43, 7999–8013. 3. Bai, S., Jain, M. K., and Berg, O. G. (2008) Contiguous binding of decylsulfate on the interface binding surface of pancreatic phospholipase A2. Biochemistry 47, 2899–2907. 4. Jain, M. K., and Berg, O. G. (2006) Coupling of the i-face and the active site of phospholipase A2 for interfacial activation. Curr. Opin. Chem. Biol. 10, 473–479. 5. Berg, O. G., Yu, B. Z., and Jain, M. K. (2009) Thermodynamic reciprocity of the inhibitor binding to the active site and the interface recognition region of IB phospholipase A2. Biochemistry 48, 3209–3218. 6. Jain, M. K., Yu, B. Z., and Berg, O. G. (1993) Relationship of interfacial equilibria to interfacial activation of phospholipase A2. Biochemistry 32, 11319–11329. [Erratum: (1994) Biochemistry 33, 8618]. 7. Berg, O. G., Rogers, J., Yu, B. Z., Yao, J., Romsted, L. S., and Jain, M. K. (1997) Thermodynamic and kinetic basis of interfacial activation: Resolution of binding and allosteric effects on pancreatic phospholipase A2 at zwitterionic interfaces. Biochemistry 36, 14512–14530. 8. Lugtigheid, R. B., Otten-Kuipers, M. A., Verheij, H. M., and De Haas, G. H. (1993) Arginine 53 is involved in head-group specificity of the active site of porcine pancreatic phospholipase A2. Eur. J. Biochem. 213, 517–522. 9. Janssen, M. J., Verheij, H. M., Slotboom, A. J., and Egmond, M. R. (1999) Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme. The effects on folding, stability and enzymatic properties. Eur. J. Biochem. 261, 197–207. 10. Kuipers, O. P., Dekker, N., Verheij, H. M., and de Haas, G. H. (1990) Activities of native and tyrosine-69 mutant phospholipases A2 on phospholipid analogues. A reevaluation of the minimal substrate requirements. Biochemistry 29, 6094–6102. 11. Kuipers, O. P., Kerver, J., van Meersbergen, J., Vis, R., Dijkman, R., Verheij, H. M., and de Haas, G. H. (1990) Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties. Protein Eng. 3, 599–603. 12. Kuipers, O. P., Vincent, M., Brochon, J. C., Verheij, H. M., de Haas, G. H., and Gallay, J. (1991) Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue. Biochemistry 30, 8771–8785. 13. van den Bergh, C. J., Bekkers, A. C., Verheij, H. M., and de Haas, G. H. (1989) Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2. Eur. J. Biochem. 182, 307–313. 14. Beiboer, S. H., Franken, P. A., Cox, R. C., and Verheij, H. M. (1995) An extended binding pocket determines the polar head group specificity of porcine pancreatic phospholipase A2. Eur. J. Biochem. 231, 747–753. 15. Kuipers, O. G., Frankan, P. A., Hendricks, R., Verheij, H. M., and De Haas, G. H. (1990) Function of the fully conserved residues Asp-99, Tyr-52 and Tyr-73 in phospholipase A2. Protein Eng. 4, 199–204. 16. Verheij, H. M., Slotboom, A. J., and de Haas, G. H. (1981) Structure and function of phospholipase A2. ReV. Physiol. Biochem. Pharmacol. 91, 91–203. 17. Yu, B. Z., Apitz-Castro, R., Jain, M. K., and Berg, O. G. (2007) Role of the 57-72 loop in specific interaction of bile salts with pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis. Biochim. Biophys. Acta 1768, 2478–2490. 18. Yuan, C., and Tsai, M. (1999) Pancreatic phospholipase A2: New views on old issues. Biochim. Biophys. Acta 1441, 215–222. 19. Ramirez, F., and Jain, M. K. (1991) Phospholipase A2 at the bilayer interface. Proteins: Struct., Funct., Genet. 9, 229–239. 20. Tsai, Y., Yu, B.-Z., Wang, Y., Chen, J. W., and Jain, M. K. (2006) Desolvation map of the i-face of phospholipase A2. Biochim. Biophys. Acta 1758, 653–665. 21. Berg, O. G., Gelb, M. H., Tsai, M. D., and Jain, M. K. (2001) Interfacial enzymology: The secreted phospholipase A2-paradigm. Chem. ReV. 101, 2613–2654. 22. Jain, M. K., Rogers, J., Jahagirdar, D. V., Marecek, J. F., and Ramirez, F. (1986) Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles. Biochim. Biophys. Acta 860, 435–447.
Yu et al. 23. Berg, O. G., Yu, B. Z., Rogers, J., and Jain, M. K. (1991) Interfacial catalysis by phospholipase A2: Determination of the interfacial kinetic rate constants. Biochemistry 30, 7283–7297. 24. Berg, O. G., and Jain, M. K. (2002) Interfacial Enzyme Kinetics, Wiley, London. 25. Piotto, M., Sanudek, V., and Sklenar, V. (1992) J. Biomol. NMR 2, 661–664. 26. Dupureur, C. M., Yu, B. Z., Jain, M. K., Noel, J. P., Deng, T., Li, Y., Byeon, I. J., and Tsai, M. D. (1992) Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73. Biochemistry 31, 6402–6413. 27. Yu, B. Z., Rogers, J., Tsai, M. D., Pidgeon, C., and Jain, M. K. (1999) Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation. Biochemistry 38, 4875–4884. 28. Thunnissen, M. M., Franken, P. A., De Haas, G. H., Drenth, J., Kalk, K. H., Verheij, H. M., and Dikstra, W. (1992) Site-directed mutagenesis of two porcine pancreatic PLA2 mutants: Y52F and Y73F. Protein Eng. 5, 597–603. 29. Thunnissen, M. M., Ab, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H., and Verheij, H. M. (1990) X-ray structure of phospholipase A2 complexed with a substrate-derived inhibitor. Nature 347, 689–691. 30. Yu, B. Z., Berg, O. G., and Jain, M. K. (1993) The divalent cation is obligatory for the binding of ligands to the catalytic site of secreted phospholipase A2. Biochemistry 32, 6485–6492. 31. Donne-Op den Kelder, G. M., De Haas, G. H., and Egmond, M. R. (1983) Localization of the second calcium ion binding site in porcine and equaine phospholipase A2. Biochemistry 22, 2470– 2478. 32. Yu, B. Z., Rogers, J., Nicol, G. R., Theopold, K. H., Seshadri, K., Vishweshwara, S., and Jain, M. K. (1998) Catalytic significance of the specificity of divalent cations as KS* and kcat* cofactors for secreted phospholipase A2. Biochemistry 37, 12576–12587. 33. Verheij, H. M., Volwerk, J. J., Jansen, E. H., Puyk, W. C., Dijkstra, B. W., Drenth, J., and de Haas, G. H. (1980) Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism. Biochemistry 19, 743– 750. 34. de Haas, G. H., Dijkman, R., van Oort, M. G., and Verger, R. (1990) Competitive inhibition of lipolytic enzymes. III. Some acylamino analogues of phospholipids are potent competitive inhibitors of porcine pancreatic phospholipase A2. Biochim. Biophys. Acta 1043, 75–82. 35. Jain, M. K., Tao, W. J., Rogers, J., Arenson, C., Eibl, H., and Yu, B. Z. (1991) Active-site-directed specific competitive inhibitors of phospholipase A2: Novel transition-state analogues. Biochemistry 30, 10256–10268. 36. Yu, L., and Dennis, E. A. (1991) Critical role of a hydrogen bond in the interaction of a phospholipase A2 with transition state and substrate analog. Proc. Natl. Acad. Sci. U.S.A. 88, 9325–9329. 37. Sekar, K., Yogavel, M., Kanaujia, S. P., Sharma, A., Velmurugan, D., Poi, M. J., Dauter, Z., and Tsai, M. D. (2006) Suggestive evidence for the involvement of the second calcium and surface loop in interfacial binding: monoclinic and trigonal crystal structures of a quadruple mutant of phospholipase A2. Acta Crystallogr. D62, 717–724. 38. Yu, B. Z., Poi, M. J., Ramagopal, U. A., Jain, R., Ramakumar, S., Berg, O. G., Tsai, M. D., Sekar, K., and Jain, M. K. (2000) Structural basis of the anionic interface preference and kcat* activation of pancreatic phospholipase A2. Biochemistry 39, 12312– 12323. 39. Rogers, J., Yu, B. Z., Tsai, M. D., Berg, O. G., and Jain, M. K. (1998) Cationic residues 53 and 56 control the anion-induced interfacial kcat* activation of pancreatic phospholipase A2. Biochemistry 37, 9549–9556. 40. Scott, D. L., and Sigler, P. B. (1994) Structure and catalytic mechanism of secretory phospholipases A2. AdV. Protein Chem. 45, 53–88. 41. Sekar, K., Kumar, A., Liu, X., Tsai, M. D., Gelb, M. H., and Sundaralingam, M. (1998) Structure of the complex of bovine pancreatic phospholipase A2 with a transition-state analogue. Acta Crystallogr. D54, 334–341. 42. Jain, M. K., and Maliwal, B. P. (1993) Spectroscopic properties of the states of pig pancreatic phospholipase A2 at interfaces and their possible molecular origin. Biochemistry 32, 11838–11846. [Erratum: (1994) Biochemistry 33, 8618].
Basis of the Interfacial Allostery of PLA2 43. Berg, O. G., Yu, B. Z., Apitz-Castro, R. J., and Jain, M. K. (2004) Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine. Biochemistry 43, 2080–2090. 44. Yu, B. Z., Polenova, T. E., Jain, M. K., and Berg, O. G. (2005) Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover. Biochim. Biophys. Acta 1712, 137–151. 45. Scott, D. L., White, S. P., Otwinowski, Z., Yuan, W., Gelb, M. H., and Sigler, P. B. (1990) Interfacial catalysis: The mechanism of phospholipase A2. Science 250, 1541–1546. 46. Jain, M. K., Yuan, W., and Gelb, M. H. (1989) Competitive inhibition of phospholipase A2 in vesicles. Biochemistry 28, 4135– 4139. 47. Jain, M. K., Yu, B. Z., Rogers, J., Gelb, M. H., Tsai, M. D., Hendrickson, E. K., and Hendrickson, H. S. (1992) Interfacial
Biochemistry, Vol. 48, No. 14, 2009 3229 catalysis by phospholipase A2: The rate-limiting step for enzymatic turnover. Biochemistry 31, 7841–7847. 48. Yuan, C., Byeon, I.-J. L., Li, Y., and Tsai, M.-D. (1999) Structural analysis of phospholipase A2 from functional perspective. 1. Functionally relevent solution structure and roles of the hydrogenbonding network. Biochemistry 38, 2909–2918. 49. Sekar, K., Eswaramoorthy, S., Jain, M. K., and Sundaralingam, M. (1997) Crystal structure of the complex of bovine pancreatic phospholipase A2 with the inhibitor 1-hexadecyl-3-(trifluoroethyl)sn-glycero-2-phosphomethanol. Biochemistry 36, 14186–14191. 50. Pan, Y. H., Epstein, T. M., Jain, M. K., and Bahnson, B. J. (2001) Five coplanar anion binding sites on one face of phospholipase A2: Relationship to interface binding. Biochemistry 40, 609–617. BI801245S
